Zernike phase contrast imaging in a TEM increases contrast of phase objects in comparison to traditional (bright field) phase imaging, leading to decreased electron irradiation dose for a desired signal-to-noise ratio. Many samples of practical interest in TEM are sensitive to electron beam irradiation because their nature changes with increasing incident electron dose, and care must be taken to avoid artefacts [2-4]. The radiation damage is of serious concern to biological samples [4] as well as to characterization of small metal particles [5].
Zernike phase contrast imaging is achieved by placing a Zernike phase plate (ZPP) in a diffraction plane (or a plane conjugated with the diffraction plane) of a TEM to provide different electron path lengths for electrons scattered by the sample with respect to electrons that were not scattered. This is performed by separating a beam of the electrons into two components that will strike the ZPP at different positions: unscattered beam strikes the phase plate at a centre of the ZPP, while the electron beams scattered by the sample strike the ZPP outside the centre. The separation of scattered and unscattered electrons is based on the angles at which they emerge from the sample: the unscattered electrons travel on a substantially direct path while the electrons scattered by the sample travel at an angle relative to optical axis of the microscope. This separation of the scattered and unscattered electrons is provided by lens action of a lens focussing the unscattered waves to a tiny point at the center of the ZPP where there is a hole, while the remainder of the beams is spread across the ZPP, and, passing through the phase plate, acquires a phase offset with respect to the unscattered waves. Traditionally a hole or a local electrostatic field is placed in the centre of the ZPP and a phase shift difference (typically ±π/2 rad) is acquired between the electrons passing through the centre of the ZPP and the beams passing outside the centre of the ZPP. The phase shift difference is induced either by thickness of the material (such as carbon) and its mean inner potential or by an applied electrostatic field in vacuum near the centre of the ZPP (referred to as Boersch or einzel lens). There are many variants of Zernike phase contrast imaging referred to as Hilbert, Chapman, Coherent Foucault imaging, etc. All of these techniques use phase plates having the center hole, and thus their phase plates are referred to herein as Zernike phase plates.
There are several problems with ZPPs that have been previously viewed as separate issues. ZPPs require a precise hole in a carbon film or a Boersch lens or einzel lens to be fabricated at the ZPP centre; then they are very difficult to align precisely on standard TEMs; and, in operation, can suffer defects due to “charging”. Due to the strict fabrication and positioning requirements they can also be expensive to manufacture and install.
Presently, ZPPs are produced by micromachining a substrate to define a Boersch or einzel lens or by using amorphous thin films with a hole of precise dimensions. In either case a hole (thin film ZPP), or a hole and a complicated electrode system (Boersch or einzel lens) is fabricated in a suitable material. Fabrication of a precise hole or electrode system a few hundred nanometers in lateral dimensions is generally challenging, and is typically performed with a focused ion beam or using micro-electro-mechanical fabrication methods. Both of these are expensive, as they require expensive equipment, and typically require multiple fabrication steps. High precision markers are typically required on the ZPP to permit alignment later on.
Boersch lens or einzel lens type ZPPs, also known as electrostatic-type ZPPs, require a complicated electrode system formed in a suitable thin film. Supporting these electrodes in alignment about a central axis with small borehole while ensuring adequate electrical isolation, and good mechanical support with a structure that minimizes interference with the electron beam in use, is challenging. These ZPPs are currently formed by micromachining (e.g. using micro-electro-mechanical systems) a substrate and deposition of conductors in a multi-step process.
For example, US2008/0035854 to Jin Jian et al. teaches a particular ZPP having a small hole and a method for microfabricating it. According to Jin Jian, phase plates are fabricated by employing several combined microfabrication techniques, including x-ray photolithography and electroplating or electroless plating.
The precise fabrication of holes in ZPPs (both thin film and electrostatic types) usually requires a focused ion beam (FIB). Unfortunately FIBs can lead to contamination of the device and damage by gallium ions. It is the hole fabrication and the need for FIB instruments that prevents many laboratories from pursuing phase contrast imaging.
Once produced, it is equally or more challenging to install the ZPP within the TEM, such that the hole is centered within the TEM so that in operation the unscattered beam passes through the hole. Typically the ZPP is positioned with piezoelectric transducers and/or sophisticated deflection equipment. It is known in the art have to place a phase plate at a back focal plane of a TEM. Unfortunately, in standard TEMs, the back focal plane of the objective lens is located in the polepiece gap, which is not wide. In some cases the polepiece gap is a fraction of a centimeter. The polepiece gap needs to accommodate the sample holder and the ZPP hardware leaving a few millimeters at most to accommodate the ZPP hardware. This is makes it all the more difficult to align and limits the ZPP hardware that can be used for positioning with respect to the electron beam.
The complications involved in this placement, as well as the great precision required for alignment has led to other suggestions that involve adding one or more transfer lenses specifically for use in the phase contrast imaging mode, such that a conjugate plane to the back focal plane is produced downstream of the objective lens. Unfortunately there are significant cost disadvantages to including the additional transfer lens, and this generally requires custom built designs, increasing dimensions of the TEM, and further substantially precludes enabling existing TEMs from using phase contrast imaging by retrofit. The transfer lens also leads to compromises in microscope performance in modes of operation other than Zernike phase plate imaging.
Once assembled and centered, ZPPs have known problems with respect to “charging”. Charging is an anomalous phase contrast transfer function observed when in phase contrast imaging mode. Jin Jian states that charging can be eliminated by coating the through-hole of the phase plate with a conductor. The anomalous phase contrast transfer function has been attributed to such processes as evaporation of contaminants from the air due to electron-beam etching, as reported in US Patent Application publication no. 2002/011,566 to Nagayama et al. Nagayama et al. teach exposure of the whole phase plate to an initial large dose of electrons, and/or keeping the phase plate at a high temperature to reduce an amount of evaporated materials on the phase plate.
There remains a need for a ZPP that is accessible with lower cost fabrication techniques, and more easily installed. Preferably the ZPP is suitable to implement and operate in a standard TEM. Furthermore, there is a need for a ZPP that is easily positioned for operation at a variety of locations within the TEM, including at a selected area aperture plane of the microscope which in some cases, is desirable.